Prostate cancer is the second most frequently diagnosed malignancy and the second leading cause of cancer-related deaths in men in the United States. Despite recent advances in the early detection and treatment of organ-confined prostate cancer, treatment results for advanced prostate cancer remain quite disappointing. In particular, current treatment modalities for androgen-independent (Al) prostate cancer only relieve the symptoms temporarily. We recently developed a prostate-restricted replicative adenovirus (PRRA), AdE1aPESEE4, to treat Al prostate cancer. Ad E1aPSESE4 replicated as efficient as a wild-type adenovirus in PSA/PSMA positive prostate cancer cells, but not in any other cell types, in tissue culture. However, the therapeutic effect of AdE1aPSESE4 in an animal tumor model was only temporary, probably due to limited tumor targeting and insufficient viral replication and distribution inside the tumor. We subsequently improved our treatment modality by integrating a expression cassette for an anti-angiogenic factor, EndoAngio (an endostatin-angiostatin fusion protein), into our PRRA to make an ant-angiogenic PRRA, EndoAngio-Ad5/35PRRA which eliminated 7 of 10 treated tumors. The remaining tumors ceased to grow in the 10-week observation period. This proposal further refines and develops our antiangiogenic PRRA therapy. Specific aim 1 will develop and validate several antiangiogenic PRRAs which express EndoAngio, sFlk1 or sTie2 to target tumor vasculature via different mechanisms. We will explore two viral backbones, Ad5 and Ad5/35 hybrid virus, to construct anti-angiogenic PRRAs. This aim will also evaluate the biodistribution and safety of antiangiogenic PRRAs after intra-tumoral and systemic administration of the virus, and assay their effects on wound healing. These steps will facilitate eventual clinical application of this therapy. Specific Aim 2 will evaluate the therapeutic advantages of antiangiogenic PRRAs over PRRA itself on s.c. prostate tumors by intra-tumor injection, prostate cancer metastases by systemic injection, and s.c. hybrid tumors composed of both PSA/PSMA-positive and negative cells by intra-tumor injection. Androgen-independent prostate cancer cells will be labeled with reporters, such as Cyan fluorescent protein, red fluorescent protein, firefly luciferase and Renila luciferase, to facilitate the detection of metastasis and analysis of therapeutic efficacy. The antiangiogenic effects of treatment on tumor vasculature will be monitored by dynamic contrast-enhanced CT (DCE-CT) imaging. We will explore dual photon fluorescent imaging to compare effects on tumor vasculature between therapies. According to our preliminary data and recent reports by others, we believe that the combination of PRRA therapy and antiangiogenic therapy presents a novel promising modality for cancer treatment and deserves more extensive investigation and further development. The study proposed in this application can be translated into the clinic to treat patients with advanced prostate cancer in the near future.